USP10/GSK3β-mediated inhibition of PTEN drives resistance to PI3K inhibitors in breast cancer
Nishi Kumari, Sarah C.E. Wright, Christopher M. Witham, Laia Monserrat, Marta Palafox, John L.C. Richard, Carlotta Costa, Moshe Elkabets, Mark Agostino, Theresa Klemm, Melissa Eccles, Alex Garnham, Ting Wu, Jonas A. Nilsson, Nikita Walz, Veena Venugopal, Anthony Cerra, Natali Vasilevski, Stephanie Bridgeman, Sona Bassi, Azad Saei, Moutaz Helal, Philipp Neundorf, Angela Riedel, Mathias Rosenfeldt, Jespal Gill, Nikolett Pahor, Oliver Hartmann, Jacky Chung, Sachdev S. Sidhu, Nina Moderau, Sudhakar Jha, Jordi Rodon, Markus E. Diefenbacher, David Komander, Violeta Serra, Pieter Johan Adam Eichhorn
Nishi Kumari, Sarah C.E. Wright, Christopher M. Witham, Laia Monserrat, Marta Palafox, John L.C. Richard, Carlotta Costa, Moshe Elkabets, Mark Agostino, Theresa Klemm, Melissa Eccles, Alex Garnham, Ting Wu, Jonas A. Nilsson, Nikita Walz, Veena Venugopal, Anthony Cerra, Natali Vasilevski, Stephanie Bridgeman, Sona Bassi, Azad Saei, Moutaz Helal, Philipp Neundorf, Angela Riedel, Mathias Rosenfeldt, Jespal Gill, Nikolett Pahor, Oliver Hartmann, Jacky Chung, Sachdev S. Sidhu, Nina Moderau, Sudhakar Jha, Jordi Rodon, Markus E. Diefenbacher, David Komander, Violeta Serra, Pieter Johan Adam Eichhorn
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Research Article Cell biology Oncology

USP10/GSK3β-mediated inhibition of PTEN drives resistance to PI3K inhibitors in breast cancer

Abstract

Activating mutations in PIK3CA, the gene encoding the catalytic p110α subunit of PI3K, are some of the most frequent genomic alterations in breast cancer. Alpelisib, a small-molecule inhibitor that targets p110α, is a recommended drug for patients with PIK3CA-mutant advanced breast cancer. However, clinical success for PI3K inhibitors (PI3Kis) has been limited by their narrow therapeutic window. The lipid phosphatase PTEN is a potent tumor suppressor and a major negative regulator of the PI3K pathway. Unsurprisingly, inactivating mutations in PTEN correlate with tumor progression and resistance to PI3K inhibition due to persistent PI3K signaling. Here, we demonstrate that PI3K inhibition leads rapidly to the inactivation of PTEN. Using a functional genetic screen, we show that this effect is mediated by a USP10-GSK3β signaling axis, in which USP10 stabilizes GSK3β, resulting in GSK3β-mediated phosphorylation of the C-terminal tail of PTEN. This phosphorylation inhibits PTEN dimerization and thus prevents its activation. Downregulation of GSK3β or USP10 resensitizes PI3Ki-resistant breast cancer models and patient-derived organoids to PI3K inhibition and induces tumor regression. Our study establishes that enhancing PTEN activity is a new strategy to treat PIK3CA mutant tumors and provides a strong rationale for pursuing USP10 inhibitors in the clinic.

Authors

Nishi Kumari, Sarah C.E. Wright, Christopher M. Witham, Laia Monserrat, Marta Palafox, John L.C. Richard, Carlotta Costa, Moshe Elkabets, Mark Agostino, Theresa Klemm, Melissa Eccles, Alex Garnham, Ting Wu, Jonas A. Nilsson, Nikita Walz, Veena Venugopal, Anthony Cerra, Natali Vasilevski, Stephanie Bridgeman, Sona Bassi, Azad Saei, Moutaz Helal, Philipp Neundorf, Angela Riedel, Mathias Rosenfeldt, Jespal Gill, Nikolett Pahor, Oliver Hartmann, Jacky Chung, Sachdev S. Sidhu, Nina Moderau, Sudhakar Jha, Jordi Rodon, Markus E. Diefenbacher, David Komander, Violeta Serra, Pieter Johan Adam Eichhorn

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Figure 2

USP10 is a regulator of PI3K signaling in breast cancer.

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USP10 is a regulator of PI3K signaling in breast cancer. (A) Immunoblot ...
(A) Immunoblot analysis of HEK293T cells expressing shRNA vectors targeting the indicated DUBs. Ctl, control. (B) Immunoblot analysis of HEK293T cells expressing USP10 shRNA vectors A and B and probed with the indicated antibodies. (C) Immunoblot analysis in HEK293T cells transfected as indicated and probed with the indicated antibodies. (D) Immunoblot analysis of MCF7 cells expressing USP10 shRNA vectors L1 and L2 probed with the indicated antibodies. Mut, mutation. (E) T47D cells expressing USP10 shRNA vectors L1 and L2, treated with IGF (300 ng/mL) as indicated. Lysates were probed with the indicated antibodies. (F and G) Immunoblot analysis of HEK293T (F) or MCF7 cells (G) or USP10 KO counterparts (USP10KO1). Lysates were probed with the indicated antibodies. (H) Overview H&E image of human breast cancer FFPE sample used for spatial transcriptomic analysis. (https://www.10xgenomics.com/datasets/human-breast-cancer-ductal-carcinoma-in-situ-invasive-carcinoma-ffpe-1-standard-1-3-0). Data were visualized using the 10x Genomics Loupe browser (version 8.0). (I) K means = 2 clustering of human breast cancer FFPE sample used to segment tumor (BRCA indicated by orange) and stromal tissue (stroma indicated by black). (J) Uniform Manifold Approximation and Projection (UMAP) analysis of cell clustering of the 2 identified clusters displaying genetically defined subclones/differential gene expression of BRCA. (K) Correlation analysis of USP10 and PIK-AKT-Hallmark gene set (HALLMARK_PI3K_AKT_MTOR-SIGNLING, M5923) in UMAP on the breast cancer tissue section. USP10 and PIK-AKT signature-only expression areas are represented in yellow and blue, respectively; co-expression is visualized in green. (L) Co-expression of USP10 and the PIK-AKT signature from UMAP (J). Inset demonstrates co-expression of USP10 and PIK-AKT-Hallmark genes in BRCA. (M) Lysates of HEK293T cells expressing recombinant UbV10 incubated with HA-tagged Ub-VS for 30 minutes. Lysates were probed with a USP10 antibody. (N) MCF7 cells expressing ubiquitin binding variant–USP10 or luciferase control, as indicated. Lysates were probed with indicated antibodies. (O) Phospholipids were isolated from cells treated with DMSO or IGF (300 ng/mL) for 1 hour, and relative PIP3 and PI(4,5)P2 levels were quantified by ELISA. *P < 0.05 by 2-tailed t test.